thermodynamic and kinetic characteristics of an α-amylase from Bacillus licheniformis SKB4

An amylolytic bacterial strain, Bacillus licheniformis SKB4 produced maximum amylase at pH 6.5 at 42 °C, and at late stationary phase (24 h) of growth. Starch and peptone were found the best supporting carbon and nitrogen source with C:N ratio of 1:2 for amylase production. The purified enzyme was non-responsive to most of the metal ions except K+ and Mg++ (1.0 mM). The enzyme was stable and active at pH 6.5. The enzyme showed optimum temperature at 90 °C with 10 min of half life (t1⁄2) at 100 °C. The Q10 of the enzyme was 1.0. The thermodynamic principles like activation energy, free energy for substrate binding and transition state of the enzyme were found 31.53, 5.53 and -17.4 KJ/Mol of starch, respectively. The kinetic constant like Vmax, Km, Kcat and catalytic efficiency (Kcat/Km) for starch were found to be 1.04 μmol mg-1 min-1, 6.2 mg ml-1, 2 × 103 S-1 and 3.22 × 102 ml mg-1 S-1, respectively. All these findings suggested that this amylase has unique characteristics for starch hydrolysis in respect to thermostability and kinetic properties. Acta Biol Szeged 58(2):147-156 (2014) Key WordS α-amylase Bacillus licheniformis thermodynamic characteristics enzyme kinetics Accepted Dec 8, 2014 *Corresponding author. E-mail: [email protected] 147 Alpha-amylase (EC 3.2.1.1, 1,4-α-D-glucan glucano hydrolase, endoamylase) hydrolyzes starch, glycogen and related polysaccharides randomly by cleaving the internal α-1,4glucosidic linkages and have great success by replacing the chemical hydrolysis of starch. α-Amylases account for ~30% of the enzyme market (Shivramakrishnan et al. 2006) and it is widely used in various industries like alcohol, brewing, sugar for liquefaction of starch; in textile industry for desizing of fabrics, etc (Gupta et al. 2003). The exploitation of amylase in world market is estimated to increase by 4% annually, significantly uses in detergents (37%), textiles (12%), starch (11%), baking (8%) and animal feed (6%) industries (Deb et al. 2013). Bacterial α-amylases, particularly from the Bacillus species are of special interest for their large-scale production under simple cultivation system and of remarkable thermostability (Prakash and Jaiswal 2010). Heat-resistant enzymes offer commercial opportunities because higher temperatures can overcome the viscosity problems of substrate (e.g. starch) and accelerated endothermic reactions (Kikani et al. 2012). Microbial enzyme production is generally subjected to the influence of different parameters of culture condition; therefore it is necessary to standardize the cultural and nutritional conditions of the organism. In industry, submerged fermentation process is generally employed for the production of microbial enzymes due to the easy control of different physico-chemical parameters, less chance of contamination and enzyme remain in the culture fluid somehow as purified form (Coronado et al. 2000; Gangadharan et al. 2006). In view of the versatile appliances of thermophilic α-amylase in different sectors, attempt has been made to optimize the submerged culture condition and characterize some relevant properties of the purified α-amylase from Bacillus licheniformis SKB4. Materials and Methods